Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method has been developed for selectively preparing the carboxyl-terminal tryptic peptide of proteins by cleavage at arginyl residues. The succinylated protein is digested with trypsin and the peptides produced are maleylated. Maleylated peptides are then submitted to cation-exchange chromatography in urea at low pH and ionic strength. Arginine-containing peptides are retained by the resin. The carboxyl-terminal peptide emerges unretarded and in pure form. This method has been applied to four proteins of known sequence. Yields as high as 88% have been obtained.
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PMID:Preparation of carboxyl-terminal tryptic peptides from proteins by cleavage at arginine. 89 87

Trypsin inhibitor was isolated from seeds of opaque-2 corn by affinity chromatography on a trypsin/Sepharose column. The two major forms of inhibitor eluted from the affinity column were separated by DEAE-cellulose chromatography in the presence of urea. One form of inhibitor is a single-chain protein that has a molecular weight of approximately 12,500; the second inhibitor has two polypeptide chains and appears to have been produced from the single-chain inhibitor by exposure to trypsin in the affinity chromatography step. The relationship of the inhibitor isolated from opaque-2 corn to an inhibitor previously isolated from an unspecified strain of maize by Hochstrasser et al. (Hochstrasser, K., Muss, M., and Werle, E. (1967) Z. Physiol. Chem. 348, 1337-1340) is discussed.
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PMID:Isolation and characterization of trypsin inhibitor from opaque-2 corn seeds. 91 65

Partial enzymatic hydrolysis of whey protein by trypsin increased solubility of this protein in water. Water-insoluble, heat-denaturated whey protein was solubilized fully by trypsinization. Optimal conditions for the enzyme reaction, established by the pH-stat technique, were: digestion at pH 8.0 and 55 C for approximately 3 h, at an enzyme-substrate ratio of 1 : 100. Under these conditions, 500 mumoles of titratable protons were liberated per g of substrate in the course of the reaction. Digestion at 40 C generated only about 400 mumoles of acid. Predenaturation of the substrate by heat did not improve digestibility. The extent of hydrolysis reached approximately 8% of all peptide bonds in the protein. Fractionation of the digest on Sephadex G-50 showed it was composed of a major fraction of highly water soluble peptides, ranging in molecular weight from approximately 500 to 5000. The gel excluded a minor fraction of larger, aggregated peptides. This aggregate was dissociated in the presence of urea and a reducing agent. All amino acids in the digest, except some lysine and arginine, were peptide bound.
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PMID:Partial enzymatic hydrolysis of whey protein by trypsin. 91 66

Flagellae of Campylobacter fetus group O, types 1, 2 and 7 were prepared. First they were separated from cell bodies using an ultramix. The suspension was then centrifuged for 20 mins. at 10000 rpm and the supernatant frozen at -40 degrees C. This is a simple method for the enrichment of preparations of flagellae, as they become tangled up and accumulate in the inferior third part of the frozen liquid. The physicochemical basis of this phenomenon was discussed. After thawing and spinning for 20 mins. at 5000 rpm, the sediment was suspended in 0.9% of NaC1. The purity of the preparation was checked by electron microscopy. Antibodies to this antigen showed no cross-reaction with O-Antigen, when tested by tube agglutination. The amino acid composition of flagellae from different O-antigen serotypes was different (see Tab. 1). Cysteine could not be detected and proline only in traces. After breakdown with urea followed by gel filtration on Sephadex G 200, breakdown products of diminishing molecular size were obtained (see Fig. 2). Discelectrophoresis after ultrasonic gave 8 zones (see Fig. 3). Irrespective of serotype, thin-layer chromatography of trypsin-hydrolysed flagellae always showed 9 ninhydrin-positive spots (see Fig. 1). Only breakdown products of ultrasonic reacted with antibody. After absorption of flagellar antibody with heterologous antigen, agglutination only occurred with homologous antigen (tab. 2-5). This showed that there were different flagellar antigens. Further experiments using immunoprecipitation demonstrated two common antigenic components, a and c, and a partially common antigenic factor bb (Fig. 4), and was the basis for a classification by three groups. The three antigenic components could be separated by gel electrophoresis and detected by immunoprecipitation (Figs. 5,6).
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PMID:[Biochemical and serological investigations of flagellae of Campylobacter fetus (author's transl)]. 93 24

Of four glycoproteins isolated from guinea pig testes, two were aspermatogenic (types I and IV) and two (types II and III) were inactive. The glycoproteins were rich in carbohydrate, varying from 41.5% to 49.5% carbohydrate by weight. Each glycoprotein had a unique amino acid composition, but in general low levels of tyrosine, tryptophan, and basic amino acids were found along with relatively high contents of serine, threonine, glutamic acid, and proline. Types I and IV glycoproteins were remarkably stable; their aspermatogenic activity was not affected by urea, trypsin, or heating at 100 degrees C in water or in 1 M HCl for 15 min. Carbohydrate analysis revealed little difference in the monosaccharide compositions of types I and IV glycoproteins, except that only the type I contained sialic acid. In contrast, types II and III glycoproteins lacked sialicacid and fucose and contained much less mannose. Both N-acetylglucosamine and N-acetylgalactosamine were present in all four glycoproteins, and they dominated in the types II and III. Fucose and at least 20-25% of the galactose appeared to occupy terminal positions in type IV glycoprotein as shown by their release after 15 min hydrolysis in 1 M HCl. All of the glycoproteins contained a relatively high percentage of galactose by weight, from 12.6 to 19.3%. The molecular weights of the glycoproteins were estimated by sodium dodecyl sulfate gel electrophoresis to be 47000, 105000 and 18000 respectively for the types I, II, and IV; type III glycoprotein showed two major bands, with molecular weights of 41500 and 22800. All the above molecular weight values are probably overestimated because of high carbohydrate content. The molecular weight of type IV glycoprotein was found to be 13000 by ultracentrifugation; a corrected value of 29000 was calculated for type I glycoprotein.
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PMID:Experimental allergic aspermatogenic orchitis. IV. Chemical properties of sperm glycoproteins isolated from guinea pig testes. 95 93

The effect of trypsin on the photosynthetic electron transport of spinach chloroplasts has been investigated by measurements of the flash-induced absorption changes, indicating chlorophyll a1 at 703 nm, chlorophyll aII at 690 nm and at 515 nm via electrochromism the electrical potential gradient across the thylakoid membrane, respectively, and of the fluorescence induction caused by moderate actinic light. It was found: (1) In the presence of benzyl viologen as electron acceptor and with water as natural electron donor trypsin, incubation leads to a complete suppression of the absorption changes of the electrochromic effect and of chlorophyll aI and chlorophyll aII. (2) Addition of System I electron donors (N-methylphenazonium sulfate plus ascorbate or 2,6-dichlorophenolindphenol plus ascorbate) fully restores the chlorophyll aI photoreaction, whereas the initial amplitude of the electrochromic absorption change at 515 nm amounts about 50% of the control value without trypsin. The chlorophyll aII inhibition remains uneffected by System I electron donors. (3) System II electron donors (benzohydroquinone plus ascorbate or TPB) are unable to overcome the inhibition of electron transport by trypsin. (4) The fluorescence induction curve in 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea-blocked chloroplasts is modified by trypsin. The level of maximal fluorescence is remarkably decreased, whereas the initial fluorescence remains constant. The rise in kinetics is slightly decelerated. From these results, it is concluded that in the linear electron transport from water to benzyl viologen, mild trypsin treatment specifically attacks System II at a site very close to the reaction center, either on the oxidizing or on the reducing side. The reaction center of System II itself is relatively stable against trypsin. Arguments are presented which argue in favor of the trypsin attack being primarily directed at the reducing side of System II.
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PMID:Studies on the nature of the inhibitory effect of trypsin on the photosynthetic electron transport of system II in spinach chloroplasts. 95 70

The enthalpies of interaction of urea with five globular proteins, ribonuclease A, trypsin, beta-lacto-globulin, ovalbumin and bovine serum albumin have been measured in aqueous solution at pH 7.0, I=0.005 M and 25 degrees C over a range of urea molality m from 0-15 mmol g-1 (where a 1 molal solution contains 1 mmol g-1). For all the proteins the interaction is exothermic, and there is an appreciable heat evolution at low urea concentrations, m less than 5 mmol g-1, which increases sharply at higher urea concentrations when the proteins undergo unfolding. If account is taken of the endothermic enthalpies of unfolding of the native proteins, the enthalpies of interactions of urea per unit mass denatured protein lie in the range -45 to -75 J g-1, corresponding to an average binding enthalpy of -23 kJ mol-1 bound urea.
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PMID:The enthalpy of interaction of urea with some globular proteins. 95 43

1. High-affinity estrogen-binding sites can be solubilized from the liver chromatin of estrogenized chickens by treatment of the chromatin with 2 M KCL/5 M urea and fractionation on hydroxylapatite. Two estrogen-binding proteins are eluted from hydroxylapatite columns by 20mM phosphate (binding protein I) and 200mMphosphate (binding protein II), respectively. 2. The binding protein I is part of a non-histone protein fraction containing acid-soluble and insoluble proteins, whereas the binding protein II elutes together with high molecular weight nonhistone proteins containing acid insoluble proteins only. Both binding proteins exhibit the smae affinity for estradiol (Kd approximately 10(-9) M). 3. From chromatin of untreated chickens very small amounts of binding protein I (0.1 pmol/mg protein compared to 1.9 pmol/mg protein from estrogenized chickens) with the smae affinity for estradiol as that from estrogenized animals can be solubilized. Binding protein II is not detectable. 4. The "soluble nuclear estrogen receptor" extracted from crude liver nucleir of estrogenized chickens by 0.5 M KCL behaves on hydroxylapatite very similarly to salt/urea-dissociated chromatin with respect to the binding protein I. No binding protein II, however, can be demonstrated. 5. Chromatography of various preparations on Bio-Gel A-1.5 m indicates that the binding protein II is a residual chromatin fragment containing an unseparated binding protein-DNA complex, whereas the binding protein I represents the solubilized nucleic-acid-free chromosomal estrogen receptor. The "soluble nuclear receptor" and the binding protein I, however, are not identical with respect to their chromatographic behaviour on Bio-Gel A-1.5m, even though their estrogen binding entity remaining after trypsin treatment seems to be very similar.
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PMID:Solubilization of the chromatin-bound estrogen receptor from chicken liver and fractionation on hydroxylapatite. 96 52

The conversion of proparathyroid hormone to parathyroid hormone (PTH) was studied in vitro employing pancreatic trypsin as a prototype converting enzyme. Digestion of intact radiolabeled bovine prohormone with trypsin (0.1%) (w/w) resulted in release of a peptide comigrating with intact hormone marker in systems resolving both on the basis of charge (urea polyacrylamide gels, pH 4.4) and size (sodium dodecyl sulfate-urea polyacrylamide gels, pH 7.2). Tryptic digestions of a synthetic analogue of bovine prohormone, ProPTH-(-6 + 34), consisting of the prohormone hexapeptide covalently bonded to the NIH2 terminus of the active fragment of the hormone, released in high yield the hexapeptide and the intact active hormone fragment before any other smaller fragments. Analyses of digestions were by: (i) thin-layer chromatography and amino acid analysis of digestion products; (ii) comparison of the biological activity of the prophormone substrate and the products of digestion; and (iii) peptide end-group analysis by the Edman method during progressive tryptic hydrolysis over 22 h. The latter experiments demonstrated cleavage of more than 75% of the hexapeptide-hormone peptide bond before cleavage of other trypsin-sensitive sites within the molecule. It is concluded that the specificity of cleavage at the hexapeptide-hormone bond in the process of intracellular hydrolysis of proparathyroid hormone resides primarily in the sequence and/or conformation of the precursor molecule; inasmuch as conversion of prohormone to hormone can be efficiently accomplished by pancreatic trypsin in vitro, there is, therefore, no need to postulate the existence of an intracellular converting enzyme within the parathyroid cell that possesses unique hydrolytic specificity.
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PMID:Conversion of proparathyroid hormone to parathyroid hormone: studies in vitro with trypsin. 99 Feb 67

A preparation of high-molecular weight kininogen (HMWK) was isolated from rabbit citrate blood plasma and purified using chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50. From 1 mg HMWK, trypsin or kallikreine of human blood plasma release 10 mkg bradykinine. The HMWK preparation is homogeneous during electrophoresis in 7.5% polyacrylamide gel in tris-glycine buffer, pH 8.3; its electrophoretic mobility corresponds to that of alpha2-globulins. The molecular weight of HMWK estimated using the collumn with Sephadex G-200, is 130.000--150.000; the sedimentation constant S20w is 7.6. Rabbit HMWK is neither a dimer, nor a trimer of low molecular weight kininogen (LMWK), since it does not degrade into subunits after treatment by 2.5% solution of sodium dodecyl sulfate, containing 8 M urea. 0.05 M 2-mercaptoethanol and 8 M urea induce HMWK splitting into 2 fragments with respective molecular weights of 80.000 and 30.000, the kinine-containing group being localized in the low-molecular weight fragment. Estimation of rates of kinine formation by different kininogenasses from highly purified HMWK and LMWK preparations showed that those kininogens are functionally different substrates, since blood plasma kallikreines release kinines from HMWK at a greater rates, whereas tissue kallikreines, e. g. human saliva kallikreine release kinines from LMWK. The specificity of kallikreines as kininogenase, to trypsin, was determined. Tripsin removes bradykinine from both kininogens at the same rates, which are an order of magnitude less than those found for kallikreines.
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PMID:[Purification and properties of high-molecular-weight rabbit kininogen]. 102 87


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